Regulation of Gene Expression, Cellular Proliferation, and Differentiation in Mammalian Development

Wai-Yee Chan, PhD, Head, Section on Developmental Genomics
Tin-Lap Lee, PhD, Staff Scientist
Eric Levens, MD, Clinical Fellow
Catherine Boucheron, PhD, Postdoctoral Fellow
Xingli Meng, MD, PhD, Postdoctoral Fellow
Vanessa Baxendale, MS, Research Associate
Shao-Ming Wu, PhD, Volunteer
Albert Hoi-Hung Cheung, MPhil, Graduate Student
Andrew J. Davis, BS, Postbaccalaureate Fellow
Diana H. Taft, BS, Postbaccalaureate Fellow
Walfred Tang, Summer Student
Jonathan Tarn, Summer Student
Darrel Young, Summer Student

Using functional genomic and systems-biological approaches to gene expression and epigenomic changes during development, we seek to understand genomic regulation of development in health and disease. At different stages of spermatogenesis, male germ cells have distinct morphological features or genetic markers that permit their easy identification and preparation as well as characterization of stage-specific gene expression and biological pathways. Such knowledge can help us (1) delineate the network of genes that regulates renewal and differentiation of germ cells and (2) develop novel methods of fertility control and aids for the infertile. Vitamin A deficiency is known to cause spermatogenic arrest at the spermatogonia stage. By studying the transcriptome of spermatogonia in the vitamin A–deficient animal, we hope to identify the elements that propel differentiation of these cells. Abnormalities of gene regulation during spermatogenesis, such as abnormal genomic methylation, lead to the development of testicular tumors. Studying changes in the epigenome of testicular tumors can help us further understand normal genetic regulation of germ cell development. To investigate selected clinical conditions, particularly those with abnormal cell growth, we applied genomic approaches to the role of mutated luteinizing hormone/choriogonadotropin receptor in testicular tumor development. Another study examines the role of mitochondria in tumorigenesis.

Regulation of Gene expression, Cell ular Proliferation, and Diff erentiation in Male Germ Cell Development

Global expression mapping of developing male germ cells

Lee, Cheung, Davis, Tarn, Baxendale, Chan W-Y; in collaboration with Rennert, Culty, Claus, Sastry

Understanding the functional significance of protein and regulatory networks requires a complete description of the cellular transcriptome. After sequencing the genes from several mammalian species, the identification of functional genes within a network is based on in silico predictions substantiated by evidence of transcription in vivo. Conservative estimates point to about 20,000 protein-encoding genes in the mammalian genome. However, in-depth analyses of the transcriptional outputs from a range of experimental approaches suggest that the information content of the genome is much more complex than previously thought. We use developing male germ cells as a model system to study important developmental processes that lead to the transition from one cell type to another. To explore and identify transcriptome complexity in male germ cells, we previously applied serial analysis of gene expression (SAGE) to profile the expression signature of the major stages of spermatogenesis, including type A spermatogonia (Spga), pachytene spermatocytes (Spcy), and round spermatids (Sptd). We identified a large number of novel transcripts, alternative splicing transcript variants, and antisense transcripts.

To provide an unbiased and higher-resolution profile of the transcriptome, we expanded our SAGE findings by incorporating whole-genome 25 bp–resolution tiling expression arrays (Affymetrix®). Using 45 million oligonucleotide probes and 35-bp probe spacing, we generated a high-definition transcriptome map of developing male germ cells with an unbiased and germ cell–specific wholegenome expression map. Preliminary data proved that combining the SAGE data set with the tiling platform is a powerful approach that provides new insights into germ cell stages. In summary, we found that over 45 percent of transcripts were not annotated; current annotation accounts for only about 30 percent of our data set, with the remainder of the data set containing mostly expressed sequence tags (EST). We identified thousands of transcript cluster units located within introns and internal exons of protein-coding genes, indicating that promoter sites are common and that transcriptional organization is complex. The transcriptional architecture implies that most genomic regions serve many functions. Although a large proportion of human transcription occurs outside the boundaries of known genes, the functional significance of such transcription remains unknown.

To facilitate further data analysis of the high-throughput genomic assay generated in our laboratory, we developed custom bioinformatics tools. GermSAGE, which was generated by SAGE, is a comprehensive web-based database of the major stages of mouse male germ cell development, with a sequence tag coverage of 150,000 in each SAGE library. It includes a total of 452,095 tags derived from Spga, Spcy, and Sptd and provides web-based tools for browsing, comparing, and searching male germ cell transcriptome data at various stages with customizable search parameters. The data may be visualized in tabulated format or further analyzed by alignment with various annotations available in the UCSC¹ Genome Browser. As a flexible platform, GermSAGE will be useful for developing a better understanding of the genetic networks that regulate spermatogonial cell renewal and differentiation and will allow novel gene discovery. It is available to the public at http://germsage.nichd.nih.gov.

We are also developing an online tool called TransfragMap to map tiling array data to various gene annotations and to mark particular gene features. The tool, which will accelerate qualitative analysis of tiling microarray data, is platform-independent and compatible with tiling microarrays from major tiling array platforms, including Affymetrix® and NimbleGen™. It will support the human and mouse genome in its initial version and will be expanded to other species.

Lee TL , Cheung HH , Claus J, Sastry C, Singh S, Vu L, Rennert OM , Chan WY. GermSAGE : a comprehensive SAGE database for transcript discovery on male germ cell development. Nucleic Acids Res 2008 [E-pub ahead of print].

Characterization of spermatogonial stem cell signaling

Lee, Chan W-Y; in collaboration with Dym, Kokkinaki

Previous work has shown that GDNF-receptor-α-1 (GFRA1) is specifically expressed in spermatogonial stem cells (SSC) and required for their stem-cell properties. To characterize the molecular phenotype of SSCs, we isolated GFRA1(+) and GFRA1(−) spermatogonia from 6-dayold mice by using magnetic-activated cell sorting with an antibody to GFRA1. In comparing microarray-based expression profiles, we found the expression of several genes to be upregulated in GFRA1(+) spermatogonia. The most overexpressed gene was Csf1r, which encodes the receptor for granulocyte-macrophage colony–stimulating factor (GM -CS F) and plays a long-established role in hematopoietic stem cell function. We also found that several chemokine ligands were highly overexpressed in SSCs. Analysis revealed the potential role of chemokine signaling in SSCs and suggested a common pathway for GFRα-1 and Csf1r, which may lead to the cells’ self-renewal.

Kokkinaki M, Lee TL , He Z, Jang J, Golestaneh N, Hofmann MC , Chan WY, Dym M. The molecular signature of spermatogonial stem cells in the 6-day-old mouse testis. Biol Reprod 2008 [E-pub ahead of print].

Characterization of mammalian RNA-dependent RNA polymerase activity

Analyses of antisense transcripts suggested the existence of RNA-dependent RNA polymerase (RdRP) activity in mouse germ cells. We identified antisense transcripts complementary to several coding exons for Tcte3, Ldh3, and Calm2. The Calm2 antisense transcript was present in mouse testis and three mouse cell lines, namely, CRL-2576 (mouse spermatogonia cell line), CRL-1715 (mouse Sertoli cell line), and CRL-6436 (mouse kidney cell line). In a knockdown experiment, we confirmed the antisense transcript as a product of the sense transcript. Specifically, we knocked down the sense transcript of Calm2 by using siRNA and demonstrated reduced levels of both sense and antisense transcripts, indicating that the synthesis of the Calm2 antisense transcript was dependent on the sense transcript. We observed that Calm2 antisense was not synthesized starting from the 3′ end of the sense mRNA, defined the sequence representing the potential start site of the action of RdRP, generated a hybrid RNA containing this sequence ligated to EGFP on its 5′ end, and introduced the hybrid into CRL-6436 cells. Orientation-specific RT-PCR2 showed production of an antisense RNA derived from hybrid RNA. The level of Calm2 antisense transcript appeared to be independent of the stage of growth of cultured cells and was ubiquitous in all mouse tissues examined (testis, ovary, liver, lung, kidney, spleen, thymus, heart, brain, and embryo). Our results provide further proof of the existence of RdRP activity in mammalian cells. Experiments to isolate and purify RdRP activity are under way.

Chan WY, Wu S, Ruszczyk LM, Lee T, Rennert OM. Antisense transcription in developing male germ cells. In: Lau YFC, Chan WY, eds. Y Chromosome and Male Germ Cell Biology in Health and Diseases. World Scientific Publishers, 2007;201-220.

Identification and functional characterization of novel spermatogenesis stage–specific genes

Tang, Lee, Tarn, Cheung, Chan W-Y; in collaboration with Rennert

Through algorithmic analysis of mouse tiling array signals of the three main cell stages of spermatogenesis with reference sequences, we identified a pool of stage-specific genes. Two of the novel genes were transmembrane and coiled-coil domain 5A (Tmco5A) and 4930563P21Rik (Tmco5B), which are expressed exclusively in mouse testis during the post-meiotic stage of male germ cell development. Both genes have a single CAGE³ tag in the testis. Comparative genomics showed conservation of the two genes in different species, including human. Evidence from protein structure prediction, gene mapping, sequence alignment, and expression profiles suggested that Tmco5A and Tmco5B likely belong to a single gene family with a specific role in male post-meiotic development or sperm function. Our studies aim to characterize the two genes in mouse testis.

Epigenomic changes in testicular cell transformation

Cheung, Lee, Davis, Taft, Young, Chan W-Y; in collaboration with Rennert

Unlike most cancers, which peak in old age, testicular germ cell tumors (TGCT) are common in young males. While genomic mutations may be one of the causes of familial TGCT, accumulating information suggests that aberrant epigenetic changes may contribute to tumorigenesis of various cancers, including TGCT. DNA methylation is one of the epigenetic hallmarks affecting chromatin structure, genomic stability, or altered transcriptional activity. Using methylated DNA immunoprecipitation and tiling array hybridization (MeDIP -chip), we compared global DNA methylation alterations between normal and testicular tumor cells. We obtained a high-resolution cytosine methylation map of the human germ cell cancer and identified more than 6,000 differential methylated regions (DM R) between normal and TGCT cells. More than 70 percent of DM Rs resided in intergenic regions, with promoter methylation accounting for 9 percent. About one-third (27 percent) of these genes demonstrated the perceived relationship between promoter methylation and gene expression, i.e., hypermethylation associated with suppression of gene expression.

A focal analysis of DM Rs located in the regulatory regions of annotated genes yielded 207 differentially methylated genes. We selected three candidate genes for further characterization in primary tumor tissues: APOLD1, RGAG1, and PCDH10. The open reading frame of APOLD1 encodes an apolipoprotein L domain–containing protein whose function is unknown. Remarkably, APOLD1 is located in 12p13.1, a TGCT -susceptibility locus identified previously by genetic linkage analysis. RGAG1 is an X-linked retrotransposon-derived neogene with unknown function. Our studies revealed that ESTs of RGAG1 were located predominantly in testis, suggesting that this retrogene might be important in male germ cell development. PCDH10 encodes a protocadherin10 membrane protein for cell adhesion, and had been implicated as a tumor suppressor gene in nasopharyngeal, esophageal, breast, colorectal, cervical, lung, and hepatocellular carcinomas. We used bisulfite sequencing and methylation-sensitive PCR to confirm differential methylation of these three genes observed in cultured tumor cells in primary testicular tumor tissues. RT-PC R confirmed differential expression of the genes in tumor and control tissues. Thus, cultured cells could be used as a model for studying the mechanism of altered methylation in tumors, and the genes may serve as novel noninvasive epigenetic markers for molecular diagnosis of TGCT.

The role of intergenic DMRs is not clear—they could simply be a consequence of inappropriate epigenetic establishment during primordial germ cell differentiation, or they may play a hidden regulatory role in the maintenance of genomic stability or chromatin condensation. Another possible function of non-genic DM Rs is the regulation of non-coding RNA s. We mapped the non-genic DMRs to a current non-coding RNA database and found that three miRNA s (miR-199a-2, miR-124a-2, and miR-184) were hypermethylated in TGCT. Notably, miR-124a was first identified in embryonic stem cells, suggesting that it may be important in differentiation. Coincidentally, it was found that miR- 124a-2 was epigenetically silenced in colon cancer, resulting in activation of the oncogene CDK6. Furthermore, we found that three snoRNAs (hb11-240, aca22, and aca8) were hypomethylated. Epigenetic changes of non-coding RNAs may lead to deregulation of genetic networks in a wider spectrum, as a single miRNA is capable of regulating several target genes.

Lee TL, Cheung AH, Davis AJ, Rennert OM, Chan WY. The paradox of DNA methylation in transcriptional control. PLoS Biol 2008;6.
Li YM, Tabatabai ZL, Lee TL, Hatakeyama S, Ohyama C, Chan WY, Looijenga LH J, Lau YFC. The Y-encoded TSPY protein: a significant marker potentially plays a role in the pathogenesis of testicular germ cell tumors. Human Pathol 2007;38:1470-1481.

Effect of vitamin A deficiency on male germ cells

Boucheron, Baxendale, Chan W-Y; in collaboration with Rennert

Vitamin A deficiency (VAD) is known to cause spermatogenesis arrest, but the molecular mechanism of VAD’s effect is not known. We plan to employ expression profiling to delineate the genetic basis of VAD-induced arrest of spermatogonial stem cell differentiation and the potential regeneration of spermatogenesis following restoration of vitamin A in the diet. We initiated a timed study of the VAD diet in which we measure the vitamin A status of the animal, the RBP⁴ level in the serum, and mRNA levels of the retinoic acid receptors RARα, RARβ, RARγ, RXRα, RXRβ, and RXRγ in the liver and testis. Given the close relationship between thyroid hormones and vitamin A metabolism, we will also study the paternally imprinted enzyme deionidase iodothyronine type II (Dio3) during VAD. Dio3 is highly expressed in developmental tissues, but its expression and function in the testis have yet to undergo study. We will begin expression profiling and epigenomic studies of the germ cells once we have established the chronology of VAD.

Biological Studies of the Luteinizing Hormone/Choriogonadotropin Receptor

The luteinizing hormone/choriogonadotropin receptor (LH/CG-R) plays a central role in human male sexual development. Mutation of LH /CG -R results in abnormal production of testosterone and disorders of sexual development. Though the role of LH /CG -R in transducing the signal of luteinizing LH/CG binding is well established, the mechanism of its action is still not fully understood. We have studied the molecular genetics of LH/CG-R in a large number of patients with activating and inactivating mutations of the receptor. Studies of these naturally occurring mutations continue to help us understand the varied disease phenotype as well as the molecular mechanism of receptor signal transduction.

Function of hCG/LH and its receptor in mammalian nervous system

Meng, Chan W-Y; in collaboration with Rennert

Individuals with activating mutations in LH/CG-R develop familial male-limited precocious puberty and often exhibit behavioral problems that may be related to dysfunction of brain cells caused by the mutated receptor. Recent studies demonstrated that human choriogonadotropin (hCG) and its receptor (LH/CG-R) may have non-gonadal functions that could be physiologically important. In the nervous system, LH/CG-R is expressed in the mammalian brain in a temporal and spatial pattern. Administration of hCG promoted nerve regeneration and neurite outgrowth in vivo and survival of primary neurons in vitro. To date, the function of hCG and its receptor in the nervous system remains unclear.

To understand the role of hCG and its receptor in the development of the mammalian nervous system, we studied the effect of the transgenes on the differentiation of the bipotent cell line PC12, which is derived from rat adrenal pheochromocytoma. Our studies showed that the differentiation effect of hCG was dependent on ligand dose and time. Both the extracellular signal–regulated kinases (ERK) and p38 mitogen–activated protein kinase (MAPK) were indispensable for the differentiation process. In addition, the phospholipase C (PLC) pathway was partially involved in hCG-induced PC12 differentiation. These findings suggest a role for hCG/LH and LH/CG-R in the neurogenesis of the mammalian nervous system. They also suggest that hCG promotes myelination by inducing myelin protein zero (P0) expression and that hCG/LH and its receptor participate in the development and maintenance of the mammalian nervous system. Thus, hCG may be a potential regeneration drug for treatment of acute neural injuries or neurodegenerative disorders.

Meng XL, Rennert OM , Chan WY. Human chorionic gonadotropin induced neuronal differentiation of PC12 cells through activation of stably expressed lutropin/choriogonadotropin receptor. Endocrinology 2007;148:5865-5873.

Molecular basis of tumorigenic activity of constitutively activated LH/CG-R mutants

Chan W-Y, Lee; in collaboration with Rennert, Leung, Su

Discovery of the presence of LH/CG-R with germline and somatic, activating mutations in patients with testicular tumor raised the question of the potential tumorigenic effect of mutated LH/CG-R. Two patients with the activating Asp578Gly mutation developed Leydig cell neoplasia; we identified a somatic, activating Asp578His mutation in several testicular tumor patients. Animal studies have so far failed to establish lines of male or female transgenic founder mice carrying LH/CG-R with the Asp578His mutation, which would reveal inherent differences between the germline and the somatic mutations. To explore the difference in the biological effects between the two mutations, we established an MA-10 Leydig cell model by stable transfection with the two mutant human receptors LH/CG-R-Asp578Gly and LH/CG-R-Asp578His. By examining gene expression profiles with cDNA microarray and using a systems-biological approach, hierarchical clustering, and multidimensional scaling analysis, we could distinguish the wild type and the two mutants. The presence of the LH/CG-R Asp578Gly mutation altered the expression of 54 genes while 49 genes were changed in the presence of the LH/CG-R Asp578His mutation. Comparing both mutants to the wild type, we found that 132 genes were differentially expressed. We also found novel regulatory pathways unique to each mutation—9 networks in LH/CG-R-Asp578Gly–expressing cells and 12 in LH/CG-RAsp578His–expressing cells. Further analyses showed that c-Myc and c-Src were the key regulators associated with Asp578Gly and Asp578His mutants, respectively. We confirmed the involvement of these two factors by molecular and functional assays. The results open a new dimension and provide a novel explanation for the role of LH/CG-R mutation in testicular tumorigenesis.

Pang AL Y, Martin MM , Martin A, Chan WY. Molecular basis of diseases of the endocrine system. In: Coleman WB, Tsongalis GJ, eds. Molecular Pathology: The Molecular Basis of Human Disease. Elsevier-Academic Press, 2009, in press.

Other Factors Aff ecting Gene Expression

Effects of mitochrondria on nuclear gene expression

Chan W-Y, Lee; in collaboration with Wong, Ma

Mitochondrial dysfunction is one of the most notable features of cancer cells. There are fewer mitochondria in rapidly growing tumor cells than in normal cells and they are smaller and exhibit a variety of altered morphologies. Somatic mutations in mitochondrial DNA are found in 30 to 100 percent of all tumors studied. The role of the altered mitochondrial genome in tumor growth is unclear. Recent studies with transmitochondrial cybrids revealed that cancer cell–derived mitochondria could alter the behavior of the host cell. To understand the genetic basis of this phenomenon, we examined the host-cell gene profile in transmitochondrial cybrids containing mitochondria derived from breast cancer cells. Preliminary results indicated distinct differences in nuclear gene expression between cybrids with normal mitochondria and cybrids with cancer cell–derived mitochrondria. We also observed marked differences in nuclear gene expression patterns between cybrids harboring various cancer cell–derived mitochrondria that behave differently with respect to tumorigenicity and various key regulators in energy metabolism, with gene signatures mimicking the breast cancer profile. To reveal the potential transcriptional regulators of the nuclear gene set (from the cybrids harboring cancer-cell derived mitochondria), we performed promoter analysis. Of the 12 known genes validated by real-time PCR, five genes demonstrated the presence of TP53 binding site while NF-kappaB binding sites were present in six genes. Four genes contain sites for both factors. Using a systems-biological analysis, we reconstructed gene networks to also confirm the potential regulation by p53 or NF-KappaB. The results suggest that mitochondria derived from breast cancer cell lines can communicate with and affect transcription activities of the nucleus. Thus, genetically altered mitochondria may actively participate in breast cancer development.

Genomic effects of naked ceria nanoparticles

Chan W-Y Lee; in collaboration with Chan S-W, Tuan

Despite the myriad applications of nanoparticles and possibly high human exposure, the impact of manufactured nanoparticles on the environment and human health is not clear. Most studies on the biological effects of nanoparticles have focused on the activities of functional components, such as drugs, proteins, antibodies, and so forth, conjugated to the nanoparticles. More recently, it has been reported that some naked nanoparticles are cytotoxic and affect cellular activities. Envirox, a trade name for naked cerium oxide (CeO₂; ceria) nanoparticles used as a diesel fuel additive, reduces fuel consumption and the emission of carbon dioxide and particulates from vehicles. However, the health impact of CeO₂ nanoparticles is poorly understood. To generate a detailed, unbiased assessment of the molecular responses to CeO₂ nanoparticles (CeO-6, 6 nm), we conducted a genomic study to identify gene expression signatures associated with nanoparticle exposure in a murine neuronal cell (HT22) model. We controlled for the effect of particle size by using 1,000 nm diameter cerium oxide (CeO-M) and for the effect of chemical property by using 300 nm diameter aluminum oxide particles (AlO). We demonstrated that cerium oxide nanoparticles induced changes in the transcriptome of murine neuronal cells—changes that depended on the chemistry and size of the nanoparticles. The genes whose expression is affected by the presence of CeO-6 are related to neurological disease, cell cycle control, and growth. Similar studies with human mesenchymal stem cells revealed the induction of osteogenic factors by CeO-6. Our observations indicate that nanoparticles are capable of inducing size- and chemistry-specific changes in cells, underscoring the need for in-depth assessment of the potential health risks of nanoparticle exposure.

Publication Related to Other Work

Ohta S, Lai EW, Morris JC, Pang ALY, Watanabe M, Yazawa H, Zhang R, Green JE, Chan WY, Sirajuddin P, Taniguchi S, Powers JF, Tischler AS, Pacak K. Metastasis-associated gene expression profile of liver and subcutaneous lesions derived from mouse pheochromocytoma cells. Mol Carcinog 2008;47:245-251.

¹University of California Santa Cruz
²Reverse transcriptase–polymerase chain reaction
³cap analysis gene expression
⁴retinol binding protein

Collaborators

Siu-Wai Chan, PhD, Columbia University, New York, NY
Janek Claus, PhD, Division of Information Technology, NICHD, Bethesda, MD
Martine Culty, PhD, Georgetown University, Washington, DC
Martin Dym, PhD, Georgetown University, Washington, DC
Maria Kokkinaki, PhD, Georgetown University, Washington, DC
Chris Y.F. Lau, PhD, University of California San Francisco, San Francisco, CA
Michael Yiu-Kwong Leung, PhD, Rush College of Medicine, Chicago, IL
Yunmin Li, PhD, University of California San Francisco, San Francisco, CA
Yewei Ma, PhD, Baylor College of Medicine, Houston, TX
Owen M. Rennert, MD, Program in Reproductive and Adult Endocrinology, NICHD, Bethesda, MD
Chandan Sastry, PhD, Division of Information Technology, NICHD, Bethesda, MD
Yan A. Su, MD, PhD, George Washington University, Washington, DC
Rocky Tuan, PhD, Cartilage Biology and Orthopaedics Branch, NIAMS, Bethesda, MD
Lee-Jun Wong, PhD, Baylor College of Medicine, Houston, TX

For further information, contact chanwy@mail.nih.gov or visit http://scg.nichd.nih.gov.